key: cord-0759498-wo5mslmf authors: Kageyama, Takahiro; Tanaka, Shigeru; Etori, Keishi; Hattori, Koto; Miyachi, Kazusa; Kasuya, Tadamichi; Iwamoto, Taro; Ikeda, Kei; Igari, Hidetoshi; Yokote, Koutaro; Nakajima, Hiroshi title: Immunological features that associate with the strength of antibody responses to BNT162b2 mRNA vaccine against SARS-CoV-2 date: 2022-02-25 journal: Vaccine DOI: 10.1016/j.vaccine.2022.02.045 sha: f0a3d00e59aeb867199c1e59ac585678f84e3fb9 doc_id: 759498 cord_uid: wo5mslmf Predictive clinical factors associated with favorable responses to BNT162b2 mRNA vaccine against SARS-CoV-2 have been reported in some studies; however, there is a subgroup with low antibody titers without well-known clinical factors reducing antibody responses. To clarify the immunological backgrounds that underlie the difference in antibody responses, we analyzed peripheral blood mononuclear cells (PBMCs) of each 20 individuals with a high anti-SARS-CoV-2 antibody titer and a low antibody titer out of 1774 healthcare workers who received BNT162b2 mRNA vaccine. A higher percentage of B cells before vaccination was associated with a higher antibody titer. Among B cells, naïve and transitional B cell frequencies were positively correlated with a higher antibody titer, whereas the frequencies of late memory B cells and plasmablasts were associated with a lower antibody titer. Fold change in the frequency of activated CD8(+) T cells upon vaccination was also correlated with high antibody titers. It has been reported that the BNT162b2 mRNA vaccine contributed to reducing the severity of COVID-19 [1] . Vaccination against SARS-CoV-2 is progressing around the world at an unprecedented rate [2] . However, the pandemic of COVID-19 has led to many SARS-CoV-2 variants, some of which have been highly transmissible and partially resistant to immune responses obtained from previous infection or vaccination [3] . Although BNT162b2 has been shown to induce vaccine-elicited neutralization against SARS-CoV-2 variants so far [4, 5] , it may be required to improve vaccines before the virus acquires critical mutations. As the humoral responses play vital roles in the protection against SARS-CoV-2 infection [6, 7] , the antibody titer status after vaccination can provide essential information to develop better vaccines and optimize vaccination strategies. We have previously reported favorable antibody responses to BNT162b2 and their predictive clinical factors in 2,015 healthcare workers [8] . Although age has been repeatedly shown to be associated with a lower antibody response among demographic factors [9, 10] , there is a subgroup with low antibody titers even in young populations without well-known factors reducing antibody responses such as taking immunosuppressive agents and glucocorticoids [8] . Therefore, we aimed to clarify the immunological backgrounds that underlie the difference in antibody responses. To address this issue, we investigated immunophenotypic characteristics in peripheral blood mononuclear cells (PBMCs), collected both before and after vaccination, among high and low responders to the BNT162b2 mRNA COVID-19 vaccine. We recruited 2,015 healthcare workers in Chiba University Hospital who received the Blood samples were obtained 0-2 weeks before the 1st dose and 2-3 weeks after the 2nd dose of vaccination. Peripheral blood mononuclear cells (PBMCs) were stored in liquid nitrogen until analysis. Anti-SARS-CoV-2S antibody was measured by Elecsys® Anti-SARS-CoV-2S on Cobas 8000 e801 module as described elsewhere [8] . PBMCs were first stained with either Zombie Green (for T cell and B cell staining panel) (Biolegend) or Zombie NIR (for monocyte staining panel) (Biolegend) to label dead cells. Then the samples were treated with Human TruStain FcX (Biolegend) to block Fc receptors. and analyzed by FlowJo software (BD). The with the antibody titer was tested using Spearman's rank correlation coefficient. P values less than 0.05 were considered statistically significant. We selected 20 young individuals with a low antibody titer after vaccination out of 1,774 healthcare workers who received 2 doses of BNT162N2 and 20 age-and sex-matched individuals with a high antibody titer. Individuals with a history of COVID-19 or a detectable antibody titer (≥0.4 U/mL) before the vaccination were excluded from the study. Individuals who were taking oral glucocorticoids or immunosuppressive medication were also excluded. We quantified the frequencies of CD4 + and CD8 + T cells, B cells, and monocytes in PBMCs, as shown in Figure S1 . Interestingly, the percentage of B cells was positively correlated with a higher antibody titer, while the percentages of T cells and monocytes were not (Figure 1 ). Among B cells, naïve and transitional B cell frequencies were positively correlated with a higher antibody titer, whereas the frequencies of late memory B cells and plasmablasts were associated with a lower antibody titer ( Figure 1 ). To our surprise, the frequencies of CD4 + T cell subsets (e.g., naïve, effector, effector memory, and central memory) were not significantly associated with antibody titers despite the well-known roles of CD4 + T cells in antibody responses. Also, there was no correlation between antibody titers and intermediate monocytes, which are important for antigen presentation. These results suggest that the abundance of naïve and immature B cells is associated with the robust antibody response against the BNT162b2 vaccine. We also analyzed the changes in cell fractions after the vaccination. The increased frequency of activated CD8 + T cells was positively correlated with a higher antibody titer (Figure 2 ). In addition, there was a trend to have higher induction of activated CD4 + T cells in high responders ( Figure 2 ). These results suggest that the robust T cells responses on the BNT162b2 vaccine are associated with high antibody responses. Recently, through the analysis of human naïve B cells, some naïve antibodies have been shown to bind to the receptor-binding domain of SARS-CoV-2 and can neutralize SARS-CoV-2 pseudoviruses even in the absence of somatic mutations [11, 12] . Furthermore, although the clinical settings are different from our study, kidney transplant recipients and dialysis patients have diminished humoral responses to BNT162b2 with reduced B cells [13] . These results suggest that the abundance of naïve B cells may be associated with the presence of a diverse BCR repertoire, which induces a good antibody response. This notion is consistent with previous findings that high antibody titers are significantly associated with younger age groups [8, 9, 10] , who are thought to have more naive B cells. Meanwhile, some studies have examined the relationship between B cell fractions and antibody production with influenza vaccines, the most widely used vaccine in humans, and have reported that naïve B cells do not correlate with high antibody responses [14, 15] . We speculate that the discrepancy between the current study on the SARS-CoV-2 vaccine and studies on influenza vaccines may be due to the presence of virus-specific memory B cells by previous infections or vaccines [16] . We also speculate that immune responses to the mRNA vaccine may differ from those to the usual inactivated vaccine. T cell activation after the vaccination is also 8 associated with high antibody titers (Figure 2 ), which may suggest broad immunogenicity of the mRNA vaccine. Further studies are required to know whether the relation between naïve B cells and the response to BNT162b2 is still observed if vaccination against COVID-19 is repeated over the years. There are several limitations to this study. The major limitation of our study is the small sample size that could be analyzed. Further studies are required to consolidate our observations. Second, the backgrounds of the subjects were not completely matched between the groups; more participants had allergic diseases in the high responder group (Table 1) . In this regard, our previous report described the association between the antibody titers and medication for allergy [8] . Unfortunately, we do not have a clear answer whether medication for allergy or the presence of allergic diseases itself is an independent factor predicting the antibody response to BNT162b2. However, there was no obvious bias in the proportion of each cell subset within the high responder group depending on the presence or absence of allergic diseases (data not shown). Also, there were significant correlations between antibody titers and the percentages of B cell subsets even when participants with allergic diseases were excluded from the analyses (data not shown). Therefore, we considered the frequencies of naïve and transitional B cells rather than allergic diseases to be a key factor in explaining the response to BNT162b2. Third, at this stage, this study did not convince the clinical importance of analyzing the specific B cell subsets prior to vaccination. However, the relationship between specific B cell subsets and favorable responses to vaccines revealed in this study may have important implications for future vaccine development. In conclusion, we have demonstrated that high proportions of naïve and transitional B cells before vaccination are associated with good responses to the BNT162b2 vaccine, although the mechanisms remain unknown. Since BNT162b2 is the first mRNA vaccine to be widely used in humans, this study has important insights into vaccine development. In order to 9 control the pandemic of COVID-19, we need to promote faster and more effective vaccination, analyzing the data obtained from ongoing vaccinations. Foundation at Chiba University. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: BNT162b2 mRNA Covid-19 Vaccine in a Nationwide Mass Vaccination Setting Impact and effectiveness of mRNA BNT162b2 vaccine against SARS-CoV-2 infections and COVID-19 cases, hospitalisations, and deaths following a nationwide vaccination campaign in Israel: an observational study using national surveillance data Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7 BNT162b2-elicited neutralization of B.1.617 and other SARS-CoV-2 variants Rapid detection of neutralizing antibodies to SARS-CoV-2 variants in post-vaccination sera Antibody Status and Incidence of SARS-CoV-2 Infection in Health Care Workers Neutralizing antibody responses to SARS-CoV-2 in symptomatic COVID-19 is persistent and critical for survival Antibody responses to BNT162b2 mRNA COVID-19 vaccine and their predictors among healthcare workers in a tertiary referral hospital in Japan Age-dependent and gender-dependent antibody responses against SARS-CoV-2 in health workers and octogenarians after vaccination with the BNT162b2 mRNA vaccine Agedependent immune response to the Biontech/Pfizer BNT162b2 COVID-19 vaccination Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients Naive human B cells engage the receptor binding domain of SARS-CoV-2, variants of concern, and related sarbecoviruses Impaired humoral immunity to SARS-CoV-2 BNT162b2 vaccine in kidney transplant recipients and dialysis patients Global analyses of human immune variation reveal baseline predictors of postvaccination responses Adjuvanted influenza-H1N1 vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events Human germinal centres engage memory and naive B cells after influenza vaccination We thank all staff in Chiba University Hospital for supporting sample collection. Low responders (n = 20)Age, year, median (IQR) 33. 5 IQR, interquartile range. SD, standard deviation.